Method of controlling ethanol production and mass production of lactic acid and transformant therefor

ABSTRACT

The present invention provides a transformant into which has been incorporated DNA for coding a foreign protein having lactate dehydrogenase activity and provided with pyruvic acid substrate affinity that equals or exceeds the pyruvic acid substrate affinity of the pyruvate decarboxylase inherent in the host organism. Said transformant can stably mass-produce lactic acid inside a host organism having the pyruvate decarboxylase gene.

TECHNICAL FIELD

The present invention relates to ethanol production control and ahigh-productivity technology for lactic acid, and more particularly to ahigh expression system suitable to lactic acid production using yeast.

BACKGROUND ART

Due to advances in recombinant DNA technology, technologies have beendeveloped that obtain the target gene product by making a foreign geneexpress itself in a host such as a microbe, mold, animal, plant, orinsect, and growing the gene's transformant. For example, by culturingyeast or the like, it is possible to produce a large volume of targetgene product through fermentation production.

There have been several attempts to produce L-lactic acid using yeast.There have also been attempts to produce L-lactic acid by incorporatinga bovine lactate dehydrogenase (LDH) gene into Saccaromyces cerevisiae(Eri Adhi et al., “Modification of metabolic pathway of Saccaromycescerevisiae by the expression of lactate dehydrogenase and deletion ofpyruvate genes for the lactic acid fermentation at low pH value,” J.Ferment. Bioeng. Vol. 86, No. 3, 284-289, 1988; Danio Porro et al.,“Development of metabolically engineered Saccaromyces cerevisiae cellsfor the production of lactic acid,” Biotechnol. Prog. Vol. 11, 294-298,1995, Kohyo (Japanese unexamined patent publication) No. 2001-516584).However, high-volume production of L-lactic acid was not observed ineither of these reports.

It is known that the pyruvate decarboxylase in Saccaromyces cerevisiaehas multiple isozymes. In ordinary yeast, pyruvate decarboxylase 1 isfunctioning. However, if this protein does not express itself due to thedestruction of the main gene, etc., pyruvate decarboxylase 5 functions,thus maintaining ethanol production (“Autoregulation of yeast pyruvatedecarboxylase gene expression requires the enzyme but not its catalyticactivity,” Ines Eberhardt, Hakan Cederberg, Haijuan Li, Stephen Koning,Frank Jordan and Stephen Hohmann, Eur. J. Biochem. Vol. 262, 191-201,1999).

DISCLOSURE OF THE INVENTION

As explained above, a technology for producing L-lactic acid in highvolume inside Saccaromyces cerevisiae has not previously been perfected.

One of the objectives of the present invention is to provide atechnology to stably produce lactic acid in high volume by controllingthe production of ethanol inside a host organism having a pyruvatedecarboxylase gene, such as Saccaromyces cerevisiae.

The inventors of the present invention conducted research focusing onthe lactate dehydrogenase (LDH) that is expressed during the productionof lactic acid inside Saccaromyces cerevisiae, and discovered that atransformant, into which has been incorporated DNA for a foreign proteinhaving lactate dehydrogenase activity and provided with pyruvic acidsubstrate affinity that equals or exceeds the pyruvic acid substrateaffinity of the pyruvate decarboxylase inherent in the host organism, iseffective for lactic acid production. Pyruvic acid is the commonsubstrate for pyruvate decarboxylase, which leads to ethanol production;lactate dehydrogenase, which acts as a catalyst for lactic acidproduction; and the like. Now, in this transformant, lactatedehydrogenase has a higher level of substrate affinity to pyruvic acidthan pyruvate decarboxylase; therefore, the ethanol production catalyzedby pyruvate decarboxylase is suppressed and the lactic acid productionby the foreign lactate dehydrogenase is promoted. In this way, it ispossible to increase lactic acid production by the transformant.

Based on the above findings, the following invention can be disclosed:

One aspect of the present invention is disclosed as a transformant, intowhich has been incorporated DNA for coding a foreign protein havinglactate dehydrogenase activity and provided with pyruvic acid substrateaffinity that equals or exceeds the pyruvic acid substrate affinity ofthe pyruvate decarboxylase inherent in the host organism. The presenttransformant also provides a transformant into which the aforementionedDNA for coding the foreign protein has been controllably incorporated bythe promoter of the pyruvate decarboxylase gene on the host chromosomeor by a homologue of said promoter that replaces said promoter.

The aforementioned foreign protein in the transformant according to thepresent invention should preferably be a bovine lactate dehydrogenase orits homologue. This protein would be especially effective if it were aprotein consisting of the amino acid sequence shown in SEQ ID NO:1, orits homologue. Furthermore, said protein should preferably be coded bythe DNA sequence shown in SEQ ID NO:3. This DNA sequence shouldpreferably be held by the transformant as the DNA sequence shown in SEQID NO:4.

The aforementioned promoter on the host chromosome should preferably bea pyruvate decarboxylase 1 gene promoter. Furthermore, this promotershould preferably use the DNA sequence shown in SEQ ID NO:2 or itshomologue.

In the present transformant, the aforementioned host organism shouldpreferably be from the Saccaromyces family and more preferably beSaccaromyces cerevisiae.

Another aspect of the present invention is disclosed as a lactic acidmanufacturing method provided with a process for culturing the presenttransformant and a process for separating lactic acid from the culturedproduct obtained in the aforementioned process. This method can stablyand efficiently manufacture lactic acid.

BRIEF EXPLANATION OF DRAWINGS

FIG. 1

FIG. 1 is a diagram illustrating the Codon Usage method used forSaccaromyces cerevisiae and its usage frequency.

FIG. 2

FIG. 2 is a diagram illustrating the homologue analysis results of thebase sequence in a bovine-derived LDH and the base sequence in amodified LDH. The top row indicates the base sequence in thebovine-derived LDH while the bottom row indicates the modified basesequence, in which the bases that are different from the base sequencein the bovine-derived LDH are indicated by symbols (A, T, C or G).

FIG. 3

FIG. 3 is a diagram illustrating the primer structure for synthesizing along-chain DNA by means of PCR used in Embodiment 1 and the synthesissteps (Steps 1 through 4).

FIG. 4

FIG. 4 is a diagram illustrating the plasmid map of the constructedvector pBTrp-PDC1-LDHKCB.

FIG. 5

FIG. 5 is a diagram illustrating a part of the process of constructingthe vector shown in FIG. 5.

FIG. 6

FIG. 6 is a diagram illustrating another part of the pBTrp-PDC1-LDHconstruction process.

FIG. 7

FIG. 7 is a diagram illustrating another part of the pBTrp-PDC1-LDHconstruction process.

FIG. 8

FIG. 8 is a diagram illustrating another part of the process (the laststep) of constructing the vector shown in FIG. 5.

FIG. 9

FIG. 9 is a diagram illustrating a structural change in the target siteof the chromosome DNA in the transformant obtained in one embodiment.

FIG. 10

FIG. 10 is a diagram illustrating the volume percentages of lactic acidand the ethanol produced in one embodiment.

FIG. 11

FIG. 11 is a diagram illustrating the trends in the volume percentagesof lactic acid and ethanol produced in one embodiment.

FIG. 12

FIG. 12 is a diagram illustrating the pyruvic acid saturation curve ofthe bovine L-lactate dehydrogenase.

FIG. 13

FIG. 13 is a diagram illustrating the Lineweaver-Burk plot of the bovineL-lactate dehydrogenase.

FIG. 14

FIG. 14 is a diagram illustrating a part of the process for constructinga vector (pBTrp-PDC1P-LDH; 7.11 kb) having a DNA segment for coding theL-lactate dehydrogenase derived from the lactic bacteriumBifidobacterium longum.

FIG. 15

FIG. 15 is a diagram illustrating another part of the process forconstructing a vector (pBTrp-PDC1P-LDH; 7.11 kb) having a DNA segmentfor coding the L-lactate dehydrogenase derived from the lactic bacteriumBifidobacterium longum; this diagram illustrates a latter stage of theprocess shown in FIG. 14.

FIG. 16

FIG. 16 is a diagram illustrating the pyruvic acid saturation curve of apyruvate decarboxylase derived from yeast.

FIG. 17

FIG. 17 is a diagram illustrating the Lineweaver-Burk plot of a pyruvatedecarboxylase derived from yeast.

PREFERRED EMBODIMENTS OF THE INVENTION

Embodiments of the present invention are explained in detail below.

(Transformant)

The foreign protein that is expressed in the transformant has lactatedehydrogenase (LDH) activity, and, the transformant has pyruvic acidsubstrate affinity that equals or exceeds the pyruvic acid substrateaffinity of this enzyme in its relationship with the pyruvatedecarboxylase inherent in the host organism that is to be transformed.

The foreign protein that is expressed in the transformant has lactatedehydrogenase (LDH) activity, and in its relationship with the pyruvatedecarboxylase inherent in the host organism that is to be transformed,has pyruvic acid substrate affinity that equals or exceeds the pyruvicacid substrate affinity of this enzyme.

Here, LDH is known as an enzyme that acts as a medium for a reactionthat produces lactic acid from pyruvic acid in the glycolytic system ofan organism such as yeast. Here, lactic acid includes L(+)-lactic acidand D(−)-lactic acid, but would preferably be L(+)-lactic acid.L(+)-lactic is produced by L(+)-LDH and D(−)-lactic acid is produced byD(−)-LDH.

LDH can be used as the foreign protein in the present invention. Varioustypes of homologues exist depending on the type of organism, or eveninside [single] organisms. In the present invention, the protein havingLDH activity includes natural LDH as well as LDH that is artificiallysynthesized through chemical synthesis or genetic engineering.

The LDH should preferably be derived from a eucaryotic microbe such asyeast; and more preferably from a higher eucaryote, such as a plant,animal, or insect; and even more preferably from even higher eucaryotes,including mammals such as bovines. Bovine-derived LDH is the mostpreferable. An example of a bovine-derived LDH is the protein consistingof the amino acid sequence shown in SEQ ID NO:1.

Bovine-derived LDH that is derived from a muscle, a heart, or the like,is available and can be used. For the enzyme number, EC 1.1.1.27 can beused.

Furthermore, the foreign protein in the present invention includeshomologues of these types of LDH. LDH homologues include proteins whichhas LDH activity, with one or several amino acids in an amino acidsequence of a naturally derived LDH replaced, void, inserted, and/oradded; and proteins which also has LDH activity that are at least 70%,and more preferably at least 80%, homologous in their amino acidsequence to a naturally derived LDH.

Note that the number of variations within the amino acid sequence is notlimited as long as the original protein function can be maintained, butshould preferably be within 70% of the total number of amino acids, morepreferably within 30%, and most preferably within 20%.

For example, a desirable homologue would be a protein with one orseveral amino acids in the amino acid sequence shown in SEQ ID NO:1replaced, void, inserted, and/or added, and which has LDH activity; or aprotein that is at least 70%, and more preferably at least 80%,homologous in its amino acid sequence to the amino acid sequence shownin SEQ ID NO:1, and which also has LDH activity.

Note that the homology in the sequence can be determined using ahomology search tool such as one available from BLAST(http://blast.genome.ad.jp/), or FASTA(http://fasta.genome.ad.jp/SIT/FASTA.html), etc.

Modification of amino acid sequence can be achieved by incorporating avariation that could be a replacement, void, an insertion, and/or anaddition as needed into the amino acid sequence that is to be modified,using a site-specific dislocation incorporation method (CurrentProtocols I Molecular Biology edit. Ausubel et al., (1987) Publish. JohnWily & Sons Selection 8.1-8.5), etc. Such a modification is not limitedto artificial incorporation or synthesis of a variation, and includesthose generated by artificial variation processes as well as thosegenerated by amino acid variations in the natural world.

The foreign protein used in the present invention should preferably havehigh substrate affinity to pyruvic acid. Here, substrate affinity meansthe Km value in the Michaelis-Menten equation as shown in Formula (1).

Mathematical Expression 1V ₀ =k ₂ E ₀ [S]/([S]+Km)  (1)

Note that V₀ is the steady-state initial speed, [S] is the substrateconcentration, [ES] is the concentration of a compound consisting of theenzyme and the substrate, E₀ is the total concentration of the enzyme,k₂ is a speed constant for ES→E+S.

The Michaelis-Menten formula is based on the relationship among thesubstrate S, the enzyme E, the substrate-enzyme compound ES, and theproduct P, expressed by Formula (2) below. k₁ is the equilibriumconstant when the product P and the enzyme E are generated from thecompound ES, and k⁻¹ is the equilibrium constant when the compound ES isdissociated into the enzyme E and the substrate S.

Mathematical  expression  2 $\begin{matrix}{{{E + S}\underset{k_{- 1}}{\overset{k_{1}}{\rightleftharpoons}}{ES}}\overset{k_{2}}{\rightarrow}{E + P}} & (2)\end{matrix}$

Here, Km can be defined from the constants using Formula (3) below.

Mathematical Expression 3V₀=k₂[ES]k ₁ [E][S]=(k ₂ +k ⁻¹)[ES]

$\begin{matrix}\begin{matrix}{\frac{\lbrack E\rbrack\lbrack S\rbrack}{\lbrack{ES}\rbrack} = \frac{k_{2} + k_{\daleth}}{k_{1}}} \\{= K_{m}}\end{matrix} & (3)\end{matrix}$

Furthermore, as indicated by Formula (4), Km can be replaced withvarious concentrations.

Mathematical Expression 4Km=(E ₀ −[ES])[S]/[ES]  (4)

Substrate affinity can be determined by determining the relationshipbetween the substrate concentration [S] and the initial speed V₀ under aconstant enzyme concentration, using a graph that shows the substrateconcentration on the horizontal axis and the initial speed on thevertical axis, for example. Substrate affinity can also be determinedfrom a Lineweaver-Burk plot.

The pyruvic acid substrate affinity of the foreign protein in thepresent invention, in its relationship with the pyruvate decarboxylaseinherent in the host organism, should preferably equal or exceed thepyruvic acid substrate affinity of this enzyme. Substrate affinityshould preferably be compared under the same temperature and pHconditions, etc. The temperature and pH conditions can be determined bytaking into consideration the environment in which these enzymescatalyze in the host organism. For example, the substrate affinity ofthese enzymes should preferably be measured under a temperature ofbetween 30 and 37° C. and a pH of between 6.0 and 7.5.

The pyruvic acid substrate affinity of the foreign protein shouldpreferably be approximately 1.5 mM or less. Because, if the pyruvic acidsubstrate affinity exceeds 1.5mM, the pyruvate decarboxylase of the hostorganism tends to react with pyruvic acid. More preferably, the valueshould be 1 mM or less. Even more preferably, the value should be 0.5 mMor less, and most preferably should be 0.1 mM or less.

Furthermore, the pyruvic acid substrate affinity of the foreign proteinshould preferably equal or exceed the pyruvic acid substrate affinity ofthe pyruvate decarboxylase of the host organism. If the pyruvic acidsubstrate affinity of the foreign is lower than the pyruvic acidsubstrate affinity of the pyruvate decarboxylase of the host organism,the pyruvate decarboxylase of the host organism tends to react withpyruvic acid. In this Specification, equal substrate affinities meansthat the corresponding substrate affinities, i.e., the Km values, areequal; a higher substrate affinity means that one substrate affinity (Kmvalue) is lower than the other corresponding Km value.

A DNA for coding such a foreign protein has been incorporated into thepresent transformant. There is no restriction on the source of this DNA,which can be a cDNA, genome DNA, synthetic DNA, or the like.

Furthermore, this DNA may possess a naturally derived base sequence forcoding LDH, or may be a DNA in which part or the whole of such a basesequence is modified and which codes a protein possessing LDH activity.It may also be a DNA possessing a naturally derived base sequence thatcodes or synthesizes an LDH homologue.

The DNA used in the present invention may possess a base sequence thatuses the Codon Usage method, which is often used for the host organismto be transformed. For example, the present DNA may possess a basesequence that has been genetically coded using the Codon Usage method inthe Saccaromyces family, especially Saccaromyces cerevisiae.

Note that a DNA can be chemically synthesized, or can be synthesized,using the method developed by Fujimoto et al. (Hideya Fujimoto,Synthetic Gene Creation Method, Plant Cell Engineering Series 7, PlantPCR Experiment Protocol, 1997, Shujunsha Co., Ltd., p 95-100), which isa known method for synthesizing long-chain DNA.

(DNA Structure)

By incorporating the DNA for coding the amino acid sequence of theforeign protein into the host organism and letting the protein coded bythis DNA express itself, it is possible to produce lactic acid in thehost cells.

For transformation, a DNA structure is used that can express a DNAsegment comprised of the present DNA inside the host cells. The form ofthe DNA structure for transformation is not limited in any way, andplasmid (DNA), bacteriopharge (DNA), retrotranspozon (DNA), or anartificial chromosome (YAC, PAC, BAC, MAC, etc.) can be selected andadopted according to the incorporation mode (inside or outside the gene)for the foreign gene or the type of host cell. Furthermore, there is nostructural restriction, such as linear or circular. Therefore, the DNAstructure can be provided with the configuration segment of any of thesevectors other than that of the present DNA itself. The preferredprocaryotic cell vectors, eucaryotic cell vectors, animal cell vectors,and plant cell vectors are well known in the field

Note that the plasmid DNA can, for example, be a YCp-typeescherichia-yeast shuttle vector, such as pRS413, pRS415, pRS416, YCp50,pAUR112, or pAUR123; a YEp-type Escherichia-yeast shuttle vector, suchas pYES32 or YEp13; a YIp-type escherichia-yeast shuttle vector, such aspRS403, pRS404, pRS405, pRS406, pAUR101, or pAUR135; a plasmid derivedfrom escherichia (a ColE-type plasmid, such as pBR322, pBR325, pUC18,pUC19, pUC119, pTV118N, pTV119N, pBluescript, pHSG298, pHSG396, orpTrc99A; a plA-type plasmid, such as pACYC177 or pACYC184; a pSC101-typeplasmid, such as pMW118, pMW119, pMW218, pMW219; or the like); or aplasmid derived from Bacillus subtilis (PUB110, pTP5, etc.). The phargeDNA can be a λ pharge (Charon4A, Charon21A, EMBL3, EMBL4, λgt100, gt11,or zap), φX174, M13mp18, or M13mp19, for example. The retrotranspozoncan be a Ty factor, for example. YAC can be pYACC2, for example.

The present DNA structure is produced by severing a fragment containing,for example, the present DNA, using an appropriate limiting enzyme, andinserting the fragment into the limiting enzyme site or multi-cloningsite of the vector DNA being used.

The first form of the present DNA structure is provided with a promotersegment that links a DNA segment comprised of the present DNA, so thatsaid DNA segment can express itself. In other words, the present DNAsegment is controllably linked by the promoter to the downstream side ofthe promoter.

It is preferable to use yeast to express the present DNA product, i.e.,a protein having LDH activity; and therefore, the use of a promoter thatexpresses itself inside yeast is preferable. For such a promoter, it ispreferable to use a pyruvate decarboxylase gene promoter, gal1 promoter,gal10 promoter, heat shock protein promoter, MF.alpha.1 promoter, PH05promoter, PGK promoter, GAP promoter, ADH promoter, or AOX1 promoter,for example. In particular, a pyruvate decarboxylase 1 gene promoterderived from the Saccharomyces family is preferable, and the use of thepyruvate decarboxylase 1 gene promoter derived from Saccharomycescerevisiae is more preferable. This is because these promoters areexpressed at high degrees in the ethanol fermentation route of theSaccharomyces family (cerevisiae). Note that said promoter sequence canbe isolated using the PCR breeding method, which uses the genome DNA ofthe pyruvate decarboxylase 1 gene of a yeast in the Saccharomyces familyas the mold. The base sequence of said promoter derived fromSaccharomyces cerevisiae is shown in SEQ ID NO:2. For the promotersegment in the present DNA structure, it is possible to use a DNAcomprised of the base sequence described in SEQ ID NO:2, as well as aDNA that is comprised of this base sequence with one or several basesvoid, replaced, or added, and which has promoter activity; or a DNA thatis hybridized with a DNA formulated from some or all of the sequences inthe base sequence shown in SEQ ID NO:2 or its complementary strand understringent conditions and which has promoter activity (in other words,the homologue of said promoter).

The second or another form of the present DNA is provided with thepresent DNA as well as a DNA segment for homologously recombining thehost chromosomes. The DNA segment for homologous recombination is a DNAsequence that is homologous to the DNA sequence in the vicinity of thetarget site in the host chromosome into which the DNA is to beincorporated. At least one, preferably two, DNA segments for homologousrecombination should be provided. For example, two DNA segments forhomologous recombination should preferably have DNA sequences homologousto the DNA on the upstream and downstream sides of the target site onthe chromosome, and the present DNA should preferably be linked betweenthese DNA segments.

When incorporating the present DNA into the host chromosome by means ofhomologous recombination, it is possible to incorporate the present DNAin a state that is controllable by a promoter on the host chromosome. Inthis case, by incorporating the present DNA, it is possible tosimultaneously destroy the endogenous gene that should have beencontrolled by said promoter and to allow the present DNA, which isforeign, to express itself instead of the endogenous gene. This processis especially useful when said promoter is a highly expressive promoterin the host cell.

To create such an expression system on the host chromosome, it ispreferable to target a high-expression gene in the host chromosome andto incorporate the present DNA into the downstream side of the promoterthat controls this gene, so that the present DNA is controlled by thepromoter. If an ethanol-fermenting microbe, such as yeast, is used asthe host, it is possible to target a pyruvate decarboxylase gene(particularly, the pyruvate decarboxylase 1 gene) and incorporate a DNAfor coding an LDH-active protein under the control of an endogenouspyruvate decarboxylase gene promoter. In this case, it is possible tomake the DNA segment for homologous recombination homologous to thesequence in the structural gene region of the LDH of the pyruvatedecarboxylase 1 gene or its vicinity (including the sequence near thestart codon, the sequence on the upstream region of the start codon, andthe sequence inside the structural gene). Preferably, an enzyme in theSaccaromyces family (especially cerevisiae) should be used as the hostand a DNA structure that targets the pyruvate decarboxylase 1 gene inthis host should be used. With such a DNA structure, the destruction ofthe pyruvate decarboxylase 1 gene and replacement of this structuralgene with LDH can be achieved using only a single vector. Because thepyruvate decarboxylase 1 gene is an enzyme that mediates theirreversible reaction from pyruvic acid to acetoaldehyde, thedestruction of its gene can be expected to suppress the production ofethanol via acetoaldehyde, and at the same time promote the productionof lactic acid by LDH, which uses pyruvic acid as the substrate.

Even the first DNA structure can be made into a DNA structure forhomologous recombination by providing it with a DNA segment forhomologous recombination with the host chromosome. In the first DNAstructure, the promoter segment inside the DNA structure can also beused as the DNA segment for homologous recombination. For example, forthe host Saccaromyces cerevisiae, a DNA structure that has the promoterin the Saccaromyces cerevisiae host chromosome, e.g., the pyruvatedecarboxylase 1 gene promoter as a promoter segment comprises atargeting vector that has said gene 1 as the target site. In this case,the first DNA structure should preferably be provided with a sequencehomologous to the structural gene region of the pyruvate decarboxylase 1gene.

Note that it is possible to link to the DNA structure, a terminator, andas needed, a cis-element such as an enhancer, a splicing signal, a polyAadditional signal, a selective marker, or a ribosome bond sequence (SDsequence). There are no special restrictions on the selective marker,and various types of known marker genes can be used, such asdrug-resistant genes or auxotrophic genes. For example, thedihydrofolate reductase gene, ampicilin-resistant gene,neomycin-resistant gene, or the like can be used.

(Transformation by DNA Structure)

Once a DNA structure has been built, it can be incorporated into anappropriate host cell by means of an appropriate method, such as thetransformation method, the transfection method, the bonding method,protoplast fusion, the electropolation method, the lipofection method,the lithium acetate method, the particle gun method, the calciumphosphate precipitation method, the agrobacterium method, the PEGmethod, or the direct microinjection method. After the incorporation ofthe DNA structure, the recipient cell is cultured in a selective media.

For the host cell, it is possible to use a bacterium such as Escherichiacoli or Bacillus subtilis; a yeast such as Saccaromyces cerevisiae,Saccaromyces pombe, or Pichia pastoris; an insect cell such as sf9 orsf21; an animal cell such as a COS cell or a Chinese hamster ovariancell (CHO cell); or a plant cell from sweet potato, tobacco, or thelike. The host cell should preferably be a microbe, such as yeast, thatcauses alcoholic fermentation, or an acid-resistant microbe, examples ofwhich include yeasts represented by the Saccaromyces family, such asSaccaromyces cerevisiae. Specific examples include the Saccaromycescerevisiae IF02260 strain and YPH strain.

In the transformant created by the DNA structure, the structuralcomponents of the DNA structure are present on the chromosomes orextrachromosomal elements (including artificial chromosomes). If the DNAstructure is maintained outside the chromosome or is incorporated intothe chromosome based on random integration, other types of yeast thatuse pyruvic acid, which is the substrate for LDH, as the substrate,e.g., the pyruvate decarboxylase gene (the pyruvate decarboxylase 1 genein the case of yeasts belonging to the Saccaromyces family), shouldpreferably be knocked out by the targeting vector.

When the DNA structure that is described above and that can achievehomologous recombination is incorporated, a DNA segment, which is thepresent DNA controllably linked by the desired promoter on the hostchromosome, is present on the downstream side of said promoter or ahomologue of said promoter that replaces said promoter. In thetransformant of a yeast in the Saccaromyces family, it is preferable toprovide the present DNA on the downstream side of the pyruvatedecarboxylase 1 gene promoter on the host chromosome or a homologue ofsaid promoter that replaces said promoter, so that the present DNA canbe controlled by said promoter.

Furthermore, a selective marker gene and part of the destroyedstructural gene (at the site corresponding to the homologous sequence onthe DNA structure) are normally present on the downstream side of thepresent DNA in a homologous recombinant.

Whether or not the present DNA has been incorporated under the desiredpromoter can be checked using the PCR method or the Southernhybridization method. For example, it is possible to check this byformulating DNA from the transformant, performing PCR with anincorporation site-specific primer, and detecting the expected band fromthe PCR product in electrophoresis. Alternatively, it is also possibleto perform PCR with a primer that has been marked with a fluorescentdye, etc. These methods are known to those skilled in the art.

(Manufacturing of Lactic Acid)

Culturing the transformant obtained by incorporating the DNA structureproduces lactic acid, which is an expression product of the foreigngene, in the cultured product. Lactic acid can then be obtained byseparating it from the cultured product. In the present invention,“cultured product” includes the culture supernatant, as well as thecultured cell or microbe, and the crushed form of the cell or microbe.

For culturing the transformant according to the present invention, theculturing conditions can be selected according to the type oftransformant. These culturing conditions are known to those skilled inthe art.

For the medium for culturing the transformant obtained using a microbe,such as Escherichia coli or yeast, as the host, any natural or syntheticmedium can be used provided that it contains a carbon source, a nitrogensource, and inorganic salts, etc. that the microbe can utilize, and thatit can efficiently culture the transformant. For the carbon source,carbohydrates such as glucose, fructose, sucrose, starch, or cellulose;organic acids such as acetic acid or propionic acid; or alcohols such asethanol or pronpanol can be used. For the nitrogen source, ammoniumsalts of inorganic or organic acids, such as ammonium chloride, ammoniumsulfide, ammonium acetate, or ammonium phosphate; othernitrogen-containing compounds; peptone; meat extract; or corn steepliquor can be used. For the inorganics, potassium phosphate, magnesiumphosphate, magnesium sulfate, sodium chloride, ferrous sulfate,manganese sulfate, copper sulfate, or calcium carbonate, etc. can beused.

Culturing is usually performed under an aerated condition, such as shakeculture or aerobic agitation culture, at 30° C. for 6 to 24 hours.During culturing, the pH level should preferably be maintained between2.0 and 6.0. The pH level can be adjusted using an inorganic or organicsalt, an alkaline solution, or the like. During culturing, anantibiotic, such as ampicilin or tetracycline, may be added to themedium as needed.

For the medium for culturing the transformant obtained using an animalcell as the host, an RPMI1640 medium or a DMEM medium, which arecommonly used, or a medium consisting of either of these with bovinefetal serum or the like added to it may be used. Culturing is normallyperformed under the presence of 5% CO₂ at 37° C. for 1 to 30 days.During culturing, an antibiotic such as kanamycin or penicillin may beadded to the medium as needed.

Culturing can be performed using a batch method or a continuous method.For the culturing method, a method can be used that obtains lactic acidin the form of a lactate, such as ammonium lactate or calcium lactate,while neutralizing the lactic acid with an alkali such as ammonium orcalcium salt. A method that obtains lactic acid as free lactic acid canalso be used.

After culturing is finished, various types of ordinary refining methodscan be used in combination for separating the lactic acid, which is agene product, from the cultured product. For example, if lactic acid isproduced inside transformed cells, the gene product can be separatedfrom the cells using a regular method, such as ultrasound destruction,grinding, or pressure crushing, to destroy the cells of the bacteria. Inthis case, protease should be added as needed. If lactic acid isproduced in the culture supernatant, solids are removed from thissolution through filtering, centrifugal separation, or the like.

For example, after the culturing process is finished, the culturedliquid can be separated into solid and liquid through at least a singlesolid-liquid separation process, such as belt pressing, centrifugalseparation, or filter pressing. It is preferable to apply a refiningprocess to the separated filtrate. In the refining process,electrodialysis, for example, can be used to remove organic acids andsugars other than lactic acid from the filtrate containing lactic acidand to obtain a lactic acid solution or ammonium lactate solution. If anammonium lactate solution is obtained, ammonium can be decomposed usinga bipolar membrane, etc. to produce an aqueous solution of lactic acidand aqueous ammonia. If the amount of organic acids and sugars otherthan lactic acid in said filtrate is small, it is also possible to skipelectrodialysis, concentrate the liquid by evaporating the moisture asneeded, and then use a bipolar membrane.

Note that for the cultured liquid and crudely extracted fractions,various refining and separation methods, such as separation andextraction using an organic solvent and distillation, can be used inaddition to the aforementioned methods to refine lactic acid or itssalt. Furthermore as needed, various types of lactic acid derivativescan be obtained by applying esterization, lactid conversion,olygomerization, or prepolymerization to the cultured liquid, crudelyextracted fraction, or its refined product. As needed, lactic acid, itssalt, and one or more kinds of lactic acid derivatives can be collectedfrom the lactic acid fermentation liquid.

Embodiments

Although embodiments of the present invention are described below, theyare not intended to specifically limit the scope of the presentinvention.

Embodiment 1 Design of the DNA Sequence of L-lactate Dehydrogenase Gene

In order to efficiently produce L-lactate dehydrogenase, which isderived from bovine, a high eucaryote, in the Saccaromyces cerevisiaefamily of yeast, we designed a new, non-naturally occurring genesequence for the DNA that codes the amino acid sequence of thebovine-derived L-lactate dehydrogenase.

-   1) The codon that is often used in Saccaromyces cerevisiae was used.-   2) Kozaks sequence (ANNATGG) was added straddling the start codon.-   3) Unstable sequences and repeated sequences in mRNA were eliminated    as much as possible.-   4) The GC content deviation was made uniform over the entire region.-   5) Measures were taken to ensure that restriction enzyme sites, not    suitable to gene cloning, would not be created inside the designed    sequence.-   6) Restriction enzyme EcoRI, XhoI, and AflIII sites, which are    useful for the two ends to be incorporated into the chromosome    incorporation type vector, were added.

For the codon usage frequency in yeast, the Saccaromyces cerevisiaecodon usage obtained from the codon usage database(http://www.kazusa.or.jp/codon/) is shown in FIG. 2. In this figure, thecodons that are used frequently for specific amino acids are identified(the underlined codons).

A new DNA sequence (999 bp) for coding a protein having the LDH activityobtained based on the design guidelines in 2) through 5) above and byapplying the frequently used codon in the codon usage shown in FIG. 1(hereafter referred to as “LDHKCB gene”) is shown in SEQ ID NO:3.Additionally, a DNA sequence (1052bp) that includes the DNA sequenceshown in SEQ ID NO:3, as well as the upstream side of its start codonand the downstream side of its stop codon (hereafter referred to as“LDHKCB sequence”), is shown in SEQ ID NO:4.

In the DNA sequence shown in SEQ ID NO:3, codons that were differentfrom those used in the original DNA sequence were used in all aminoacids except methionine. Note that the newly adopted codons were allfrequently used codons from among those shown in FIG. 1. Furthermore,FIG. 2 shows the result of a computer-based homology analysis of theoriginal bovine-derived LDH gene and the LDHKCB gene. As is clear fromFIG. 2, it was discovered that a large number of replacements wereneeded over almost the entire DNA sequence.

Embodiment 2 Complete Synthesis of the LDHKCB Sequence

In this embodiment, the method of Fujimoto et al. was used, which is aknown method for synthesizing long-chain DNA (Hideya Fujimoto, SyntheticGene Creation Method, Plant Cell Engineering Series 7, Plant PCRExperiment Protocol, 1997, Shujunsha Co., Ltd., p95-100). The principlebehind this method is as follows: oligonucleotide primers of around 100mer are created so as to have an overlap of between 10 and 12 mer at the3′ end. Then, the missing area is extended using the overlapped regionbetween the oligonucleotide primers, and is amplified through a PCRusing the primers at both ends. This operation is sequentially repeatedto synthesize the target long-chain DNA. For the PCR amplifier, Gene AmpPCR system 9700 (P.E. Applied Biosystems Inc.) was used.

Specifically, the two kinds of oligonucleotide primers to be linked weremixed first, and a DNA extension reaction was carried out at 96° C. for2 minutes, 68° C. for 2 minutes, 54° C. for 2 minutes, and 72° C. for 30minutes under the presence of KOD-plus-DNA polymerase (Toyobo). Next,using 1/10 of the sample as a mold, a PCR (polymerase chain reaction)was carried out. In this reaction, the sample was first kept at 96° C.for 2 minutes; it was then put through 25 cycles (with each cycleconsisting of 96° C. for 30 seconds, 55° C. for 30 seconds, and 72° C.for 90 seconds) under the presence of primers at both ends; and finallywas kept at 4° C. For the reaction, the buffer and dNTPmix, etc. thatcame with the DNA polymerase were used.

A series of overlapping PCRs were carried out according to FIG. 3 tocreate the gene fragments that were ultimately targeted. The DNAsequences of all 28 primers shown in FIG. 3 (BA, B01, BB, B02, BC, B03,BD, B04, BE, B05, BF, B06, BG, B07, BH, B08, BI, B09, BJ, B10, BK, B11,BL, B12, BM, B13, BN, and B14) are shown in SEQ ID NOS:5 through 32,respectively. After the base sequence of the synthesized LDHKCB gene wasverified, a restriction enzyme process using EcoRI was applied. Thesequence was then linked to the pCR2.1 TOPO Vector (Invitirogen), towhich an enzyme process using EcoRI had been applied in a similarmanner, using a normal method. This vector is referred to as thepBTOPO-LDHKCB vector.

Embodiment 3 Construction of a Vector for Yeast Chromosome Incorporation

A yeast chromosome incorporation-type vector was constructed using theLDHKCB sequence completely synthesized in Embodiment 2. This vector isreferred to as pBTRP-PDC1-LDHKCB and its plasmid map is shown in FIG. 4.

1. Isolating PDC1P Fragments for Constructing pBTrp-PDC1-LDHKCB

PDC1P fragments were isolated by means of the PCR amplification methodthat uses the genome DNA of the Saccaromyces cerevisiae YPH strain(Stratagene Corp.) as the mold.

The genome DNA of the Saccaromyces cerevisiae YPH strain was preparedusing the Fast DNA Kit (Bio 101 Inc.), which is a genome preparationkit, and according to the detailed protocol described in the appendix.DNA concentration was measured using the spectrophotometer Ultro spec3000 (Amersham Pharmacia Biotech Inc.).

For the PCR, Pyrobest DNA Polymerase (Takara Shuzo Co., Ltd.), which issaid to produce highly precise amplified fragments, was used as theamplification enzyme. A total of 50 μl of reaction solution, containinga 50 ng/sample of the genome DNA of the Saccaromyces cerevisiae YPHstrain prepared using the aforementioned method, a 50 pmol/sample of theprimer DNA, and a 0.2 units/sample of Pyrobest DNA Polymerase, wereprepared. The reaction solution was put through DNA amplification usinga PCR amplifier system (Gene Amp PCR system 9700 made by P.E. AppliedBiosystems Inc.). The reaction conditions for the PCR amplifier were asfollows: the solution was first kept at 96° C. for 2 minutes; it wasthen put through 25 cycles (with each cycle consisting of 96° C. for 30seconds, 55° C. for 30 seconds, and 72° C. for 90 seconds); and finallywas kept at 4° C. The PCR-amplified fragments were verified to beamplified gene fragments by means of electrophoresis in 1% TBE agarosegel. Note that a synthetic DNA (Sawaday Technology Co.) was used as theprimer DNA for the reaction; the DNA sequence of this primer isdescribed below.

A restriction enzyme BamH1 site was added to the PDC1P-LDH-U (31 mer, Tmvalue of 58.3° C.) end.

(SEQ ID NO: 33) ATA TAT GGA TCC GCG TTT ATT TAC CTA TCT C

A restriction enzyme EcoRI site was added to the PDC1P-LDH-D (31 mer, Tmvalue of 54.4° C.) end.

(SEQ ID NO: 34) ATA TAT GAA TTC TTT GAT TGA TTT GAC TGT G2. Constructing a Recombinant Vector Containing a Promoter and theTarget Gene

Under the control of the promoter sequence for pyruvate decarboxylase 1gene (PDC1) derived from Saccaromyces cerevisiae, a bovine-derivedL-lactate dehydrogenase gene (LDH gene) was used as the target gene.

The new chromosome incorporation-type vector constructed forconstructing the present recombinant vector was named pBTrp-PDC1-LDHKCB.The details of an example of constructing the present vector aredescribed below. An overview of the present embodiment is illustrated inFIGS. 5 through 8. Note that the procedure for constructing the vectoris not limited to that described here.

When constructing the vector, the PDC1 gene promoter fragment (PDC1P)971 bp and the PDC1 gene downstream region fragment (PDC1D) 518 bp wereisolated using the PCR amplification method that uses the genome DNA ofthe Saccaromyces cerevisiae YPH strain as the mold, as described above.Although the procedure described above was used for PCR amplification,the following primers were used for amplifying the PDC 1 gene downstreamregion fragment:

A restriction enzyme Xhol site was added to the PDC1D-LDH-U (34 mer, Tmvalue of 55.3° C.) end.

(SEQ ID NO: 35) ATA TAT CTC GAG GCC AGC TAA CTT CTT GGT CGA C

A restriction enzyme Apal site was added to the PDC1D-LDH-D (31 mer, Tmvalue of 54. 4° C.) end.

(SEQ ID NO: 36) ATA TAT GAA TTC TTT GAT TGA TTT GAC TGT G

After the PDC1P and PDC1D amplified gene fragments obtained through theaforementioned reactions were each refined through an ethanolprecipitation process, the PDC1P amplified fragment and the PDC1Damplified fragment were reacted with restriction enzymes BamHI/EcoRI andXhoI/ApaI, respectively. Note that all of the enzymes used below weremade by Takara Shuzo Co., Ltd. Additionally, the series of detailedoperations in the ethanol precipitation process and the restrictionenzyme processes were based on “Molecular Cloning—A Laboratory Manual,Second Edition” (Maniatis et al., Cold Spring Harbor Laboratory Press,1989).

The series of reaction operations for constructing the vector werecarried out according to a generally known DNA subcloning method. Thatis, the PDC1P fragment, which had been amplified by the aforementionedPCR method and to which a restriction enzyme process had been applied,was linked through a T4 DNA Ligase reaction to the pBluescriptIISK+vector (Toyobo), to which the restriction enzyme BamHI/EcoRI (TakaraShuzo) and the dephosphorylase Alkaline Phosphotase (BAP, Takara Shuzo)had been applied (FIG. 5 top row). For the T4 DNA Ligase reaction, theLigaFast Rapid DNA Ligation System (Promega Corporation) was used,following the included detailed protocols.

Next, transformation to competent cells was carried out using thesolution in which the ligation reaction had taken place. For thecompetent cells, Escherichia coli JM109 strain (Toyobo) was used,following the included detailed protocols. The obtained culturedsolution was sprayed onto an LB plate containing the antibioticampicilin at the rate of 100 μg/ml, and incubated overnight. The colonythat grew was verified with the colony PCR method using the primer DNAof the insert fragment, and with a restriction enzyme process for aplasmid DNA solution prepared through miniprep. The target vector(pBPDC1P vector) was then isolated (FIG. 5 middle row).

Next, as shown in the middle row in FIG. 5, the LDHKCB gene fragment,obtained by processing the pBTOPO-LDHKCB vector constructed by KabushikiKaisha Toyota Chuo Kenkyusho with the restriction enzyme EcoRI and theend-modification enzyme T4 DNA polymerase, was subcloned, using the sameoperation as described above, into the pPDC1P vector likewise processedwith the restriction enzyme EcoRI and the terminal modification enzymeT4 DNA polymerase, to create the pBPDC1P-LDHKCB vector (FIG. 5 bottomrow).

On the other hand, as shown in FIG. 6, the LDH gene (derived fromBifidobacterium longum) fragment obtained by processing the pYLD1vector, constructed by Toyota Jidosha Kabushiki Kaisha, with therestriction enzyme EcoRI/AatII and the end-modification enzyme T4 DNApolymerase, was subcloned, using the same operation as described above,into the pBPDC1P vector likewise processed with the restriction enzymeEcoRI and the terminal modification enzyme T4 DNA polymerase, to createthe pBPDC1P-LDH1 vector (FIG. 6). Note that the aforementioned pYLD1vector has been incorporated into Escherichia coli (name: E. coli pYLD1)and has been internationally deposited with the International PatentOrganism Depositary of the National Institute of Advanced IndustrialScience and Technology (Chuo No. 6, 1-1-1 Higashi, Tsukuba-shi,Ibaraki-ken, 305-8566 Japan) under deposit No. FERM BP-7423 inaccordance with the Budapest Convention (the original deposit date: Oct.26, 1999).

Next, as shown in FIG. 7, this vector was processed with XhoI/ApaI andwas linked to the amplified PDC1D fragment, to which a restrictionenzyme had been applied in the same manner, to create the pBPDC1P-LDHvector (FIG. 7 top row). Lastly, the pBPDC1P-LDHII vector, to which aEcoRV process had been applied, was linked to a Trp maker fragmentobtained by applying the AatII/SspI process and the T4 DNA polymeraseprocess to the pRS404 vector (Stratagene Inc.), to construct thepBTrp-PDC1-LDH vector.

Next, as shown in FIG. 8, the pBPDC1P-LDHKCB vector was processed withrestriction enzymes ApaI/EcoRI; separately, the pBTrp-PDC1-LDH vectorwas processed into fragments containing the Trp marker processes withrestriction enzymes ApaI and StuI. The amplified fragments were linkedto construct the chromosome incorporation-type pBTrp-PDC1-LDHKCB vector,which is the final construct.

In order to verify the constructed chromosome incorporation-typepBTrp-PDC1-LDHKCB vector, base sequence determination was performed. AnABI PRISM 310 Genetic Analyzer (P.E. Applied Biosystems Inc.) was usedfor analyzing the base sequence, and details such as the method ofpreparing samples and using the instrument were based on the manualprovided with the analyzer. The vector DNA to be used as the sample wasprepared with an alkali extraction method. Before using this sample, itwas first column-refined using the GFX DNA Purification kit (AmershamPharmacia Biotech Inc.), and then its DNA concentration was measuredwith the Ultro spec 3000 spectrophotometer (Amersham Pharmacia BiotechInc.).

Embodiment 4 Transformation of Yeast

The gene incorporates into yeast according to the method developed byIto et al. (Ito, H., Y. Fukuda, K. Murata and A. Kimura, Transformationof intact yeast cells treated with alkalications, J. Bacteriol. Vol.153, p 163-168). That is, the yeast IF02260 strain (a strain registeredwith the Institute of Fermentation, Osaka), from which thetryptophan-synthesizing function had been removed, was cultured in 10 mlYPD medium at 30° C. until it reached the logarithmic growth phase. Itwas then collected and rinsed with TE buffer. Next, 0.5 ml of TE bufferand 0.5 ml of 0.2M lithium acetate were added, and the mixture wasshake-cultured at 30° C. for 1 hour. Then, the chromosomeincorporation-type pBTrp-PDC1-LDHKCB vector constructed using the methodin Embodiment 3 was processed with restriction enzymes ApaI and SacI(both made by Takara Shuzo), and was added to the culture.

After the present yeast suspension was shake-cultured at 30° C. for 30minutes, 150 μl of 70% polyethylene glycol 4000 (Wako Pure ChemicalIndustries) was added and the mixture was agitated well. Then, after themixture was shake-cultured at 30° C. for 1 additional hour, a heat shockwas applied at 42° C. for 5 minutes. The yeast was then rinsed andsuspended in 200 μl of water, and this solution was applied to atryptophan selective culture medium.

The obtained colony was applied to a new tryptophan streak culturemedium, and selected strains that had demonstrated stability werechecked for gene incorporation using PCR analysis. The genome DNA of theyeast to be used for PCR was prepared by shake-culturing a single colonyin 2 ml of YPHD media overnight, collecting the yeast, adding 50 mMTris-HCL 500 μl and glass beads (425-600 μm, acid washed, SIGMA), andputting the mixture through a vortex at 4° C. for 15 minutes. Thesupernatant of this solution was put through ethanol precipitation andwas dissolved in 50 μl of sterilized water. Using 5 μl of the preparedgenome DNA as the mold, PCR was performed in 50 μl of reaction solution.EX Taq DNA Polymerase (Takara Shuzo) was used for the DNA amplificationenzyme, and the PCR amplifier Gene Amp PCR system 9700 (P.E. AppliedBiosystems Inc.) was used. The reaction condition for the PCR amplifierwas as follows: the solution was first kept at 96° C. for 2 minutes; itwas then put through 30 cycles (with each cycle consisting of 96° C. for30 seconds, 55° C. for 30 seconds, and 72° C. for 90 seconds); andfinally was kept at 4° C. The sequences of the primers used were asfollows:

(Sequence number 37) LDH-KCB-U : TGG TTG ATG TTA TGG AAG AT (20 mer)(Sequence number 38) LDH-KCB-D : GAC AAG GTA CAT AAA ACC CAG (21 mer)(Sequence number 39) PDC1P-U3 : GTA ATA AAC ACA CCC CGC G (19 mer)

Those strains that possess a stable tryptophan-synthesizing function andfor which PCR was verified under these primers were judged to betransformants into which the LDHKCB gene had been appropriatelyincorporated. In the present embodiment, three kinds of yeast strains,namely KCB-27, KCB-210, and KCB-211, were obtained as suchtransformants. FIG. 9 shows the genome chromosome structures of theseyeast Saccaromyces cerevisiae transformants.

Embodiment 5 Measurement of L-lactic Acid Production Volume in theTransformants

Fermentation experiments were conducted on the three kinds oftransformants created. As pre-culturing, the transformants were culturedovernight in a YPD solution medium with 2% glucose concentration. Afterthe yeast were collected and rinsed, they were planted in a YPD solutionmedium with 15% glucose concentration such that the yeast concentrationwas 1% (0.5 g/50 ml), and were left to ferment at 30° C. for severaldays. This fermented solution was sampled every 24 hours and the volumeof L-lactic acid contained was estimated[sic]. For measuring the volumeproduced, Biosensor BF-4 (Oji Scientific Instruments) was used followingthe detailed measurement methods described in the user's manual for theinstrument.

FIG. 10 and Table 1 show the measurement results of the L-lactic acidvolume and the ethanol volume in the cultured solution on the third dayof fermentation with a glucose concentration of 15%. As for the KCB-27strain, FIG. 11 and Table 2 show the trends in L-lactic acid volume andethanol volume between the 0^(th) and 5^(th) days.

TABLE 1 Production volume (%) Strain Lactic acid Ethanol IF02260 0 7.34KCB-27 4.92 5.01 KCB-210 4.78 5.14 KCB-211 4.57 5.26

TABLE 2 Number of Production days passed Strain volume (%) 0 1 3 5IF02260 Glucose 15 1.84 0 0 Lactic acid 0 0 0 0 Ethanol 0 6.69 7.21 7.38KCB-27 Glucose 15 2.2 0 0 Lactic acid 0 3.01 4.73 4.92 Ethanol 0 4.695.11 5.06

Even though only a single copy of the LDHKCB gene had been incorporatedinto each of the transformants into which the LDHKCB gene wasincorporated (KCB-27, KCB-210, and KCB-211 strains), production ofbetween 4.5 and 5.0% (45.0 to 50.0 g) of L-lactic acid was confirmed ineach 1 L of the culture solution. Meanwhile, ethanol production was 5%,which was approximately 2.5% less than would be expected with the parentstrain.

This result clearly indicates an extremely significant increase in theproduction of L-lactic acid in Saccaromyces cerevisiae as compared tothe cases reported in the past. Furthermore, this increase in productionvolume is presumed to be due to the introduction of LDH with substrateaffinity equal to or higher than the pyruvic acid substrate affinity ofthe pyruvate decarboxylase derived from yeast. It is also presumed thatintroducing LDH under the control of the Pyruvate decarboxylase 1 genepromoter on the chromosome contributed as well.

The above result shows that even a single copy can produce a significantincrease in the production volume. Therefore, it can be presumed thatthe introduction of two or more copies would increase the productionvolume even further.

Embodiment 6 Measurement of the Pyruvic Acid Substrate Affinity(Michaelis Constant and Km Value) of the L-lactate Dehydrogenase Derivedfrom Various Eucaryotes

The pyruvic acid substrate affinity (Michaelis constant and Km value) ofthe L-lactate dehydrogenase derived from bovine was measured using themethod described below. The result was compared to the pyruvic acidsubstrate affinity of the L-lactate dehydrogenase derived fromLactobacillus.

To measure the pyruvic acid substrate affinity (Km value), pyruvic acid,NADH, and FBP were first added to refined L-lactate dehydrogenase tocause a fermentation reaction. Then, the reduction rate of NADH wasdetermined with a spectrophotometer (Ubest-55 made by JASCO) at ABS: 340nm, and the activity of each L-lactate dehydrogenase was determined.

Next, based on the value of the L-lactate dehydrogenase activity atvarious pyruvic acid concentration levels, a Lineweaver-Burk plot wascreated computed from the pyruvic acid saturation curve and theindividual inverse numbers. The pyruvic acid substrate affinity(Michaelis constant and Km value) of the L-lactate dehydrogenase wasthen determined.

A solution prepared by adding 50 mM MOPS buffering solution (made byNarakai) with its pH adjusted to 7.0, 0.15 mM NADH (made by SIGMA), and1 mM FBP (SIGMA) to 0.05 μg of refined bovine muscle-derived L-lactatedehydrogenase (SIGMA) was kept at 37° C. for 2 to 3 minutes.

Next, pyruvic acid (Wako Pure Chemical Industries) at variousconcentration levels (0.01 to 100 mM) was added. After quick mixing, thesolution was set in a spectrophotometer (Ubest-55 made by JASCO) and thechange in absorbance over 1 minute was determined at ABS: 340 nm.

Note that the LDH activity was determined using the following formula(Mathematical expression 5):

[Mathematical Expression 5]

$\begin{matrix}{{{LDH}\mspace{14mu}{activity}\mspace{14mu}{value}\mspace{11mu}\left( {U\text{/}\;{mg}\mspace{14mu}{protein}} \right)} = {\frac{{Absorbance}\mspace{14mu}{change}\mspace{14mu}{over}\mspace{14mu} 1\mspace{14mu}{minute}}{0.0063} \times \frac{1000\mspace{11mu}\mu\; g}{0.05\mu\; g} \times \frac{200\mspace{11mu}\mu\; l}{1000\mspace{11mu}\mu\; l} \times \frac{1}{1000}}} & \left\lbrack {{Mathematical}\mspace{14mu}{expression}\mspace{14mu} 5} \right\rbrack\end{matrix}$

Note that the operation was carried out in accordance with 1) Minowa T.,Iwata S., Sakai H., and Ohta T., Sequence and characteristics of theBifidobacterium longum gene encoding L-lactate dehydrogenase and primarystructure of the enzyme; a new feature of the allosteric site., Gene,1989, Vol. 85, 161-168; and 2) the description in the user's manualincluded with SIGMA Lactate Dehydrogenase (LDH/LD).

Based on the LDH activity obtained, a pyruvic acid saturation curve wascreated with [V]=LDH enzyme accuracy (U/mg) on the vertical axis and[S]=pyruvic acid concentration (mM) on the horizontal axis. This graphis shown in FIG. 12.

Furthermore, a Lineweaver-Burk plot computed from individual inversenumbers was respectively created with 1/[V] and 1/[S] on the verticaland horizontal axes. FIG. 13 shows this plot for the present enzyme.

The pyruvic acid concentration substrate affinity (Km value) wascomputed utilizing the fact that the intersection between theLineweaver-Burk plot and the 1/[V] axis is 1/Vmax and the intersectionbetween the plot and the 1/[S] axis is −1/Km.

Each Lineweaver-Burk plot showed that the pyruvic acid substrateaffinity of the L-lactate dehydrogenase derived from bovine muscle was0.1 mM. The inventors of the present invention possess the knowledgethat the pyruvic acid substrate affinity of the L-lactate dehydrogenasederived from the Lactobacillus Bifidobacterium longum is 1.0 mM.

The volumes of L-lactic acid and alcohol produced by a recombinant yeastobtained by incorporating the DNA for coding the L-lactate dehydrogenasederived from the Lactobacillus Bifidobacterium longum into a host andletting it express itself to a high degree are described below. (Thefermentation conditions were 30° C. for 3 days in a YPD medium with 15%glucose concentration.) FIGS. 14 and 15 show the vector(pBTrp-PDC1P-LDH; 7.11 kb) for obtaining said Lactobacillus-derivedrecombinant yeast and the process for constructing it. In saidtransformed yeast, said gene was controllably incorporated by the PDC1promoter through knock-in into the PDC gene locus on the hostchromosome. It has been confirmed that this transformed yeast is a2-copy body into which said L-lactate dehydrogenase genes have beenincorporated into a pair of PDC gene loci on the host chromosome. Notethat the operations for constructing said vector and transforming theyeast are the same as those in Embodiments 3 and 4, except for thosesteps shown in FIGS. 14 and 15.

As shown in Table 3, when this result is compared with the resultobtained from the bovine muscle-derived L-lactate dehydrogenase testedin Embodiment 5, the volume of L-lactic acid produced by the yeast withthe bovine muscle-derived LDH incorporated is approximately twice thatof the yeast with the Lactobacillus-derived LDH incorporated.

TABLE 3 Production volume (%) L-lactic acid Ethanol Yeast with bovinemuscle-derived LDH 4.92% 5.01% incorporated (pyruvic acid substrateaffinity: 0.1 mM) Yeast with Lactobacillus Bifidobacterium 2.46% 6.11%longum-derived LDH incorporated (pyruvic acid substrate affinity: 1.0mM)

Embodiment 7 Measurement of the Pyruvic Acid Substrate Affinity(Michaelis Constant and Km Value) of the Pyruvate Decarboxylase Derivedfrom Yeast

To measure the activity of the yeast-derived pyruvate decarboxylase andthe pyruvic acid substrate affinity (Km value), pyruvic acid, NADH, andthiamin were first added to refined pyruvate decarboxylase to cause afermentation reaction. Then, the reduction rate of NADH was determinedwith a spectrophotometer (Ubest-55 made by JASCO) at ABS: 340 nm, andthe activity of the yeast-derived pyruvate decarboxylase was determined.

Next, based on the value of the pyruvate decarboxylase activity atvarious pyruvic acid concentration levels, a Lineweaver-Burk plotcomputed from the pyruvic acid saturation curve and the individualinverse numbers was created, and the pyruvic acid substrate affinity(Michaelis constant and Km value) of the pyruvate decarboxylase wasdetermined.

A solution prepared by adding 34 mM Imidasol HCl buffering solution(made by Wako Pure Chemical Industries) with its pH adjusted to 7.0,0.15 mM NADH (made by SIGMA), and 0.2 mM Tiamine Pyrophospate (SIGMA) to0.05 μg of refined yeast-derived pyruvate decarboxylase (SIGMA) was keptat 37° C. for 2 to 3 minutes.

Next, pyruvic acid (Wako Pure Chemical Industries) at variousconcentration levels (0.01 to 100 mM) was added. After quick mixing, thesolution was set in a spectrophotometer (Ubest-55 made by JASCO) and thechange in absorbance over 1 minute was determined at ABS: 340 nm.

Note that the PDC activity was determined using the following formula(Mathematical expression 6):

[Mathematical Expression 6]

$\begin{matrix}{{{PDC}\mspace{14mu}{activity}\mspace{14mu}{value}\mspace{11mu}\left( {U\text{/}\;{mg}\mspace{14mu}{protein}} \right)} = {\frac{{Absorbance}\mspace{14mu}{change}\mspace{14mu}{over}\mspace{14mu} 1\mspace{14mu}{minute}}{0.0063} \times \frac{1000\mspace{11mu}\mu\; g}{0.05\mu\; g} \times \frac{200\mspace{11mu}\mu\; l}{1000\mspace{11mu}\mu\; l} \times \frac{1}{1000}}} & \left\lbrack {{Mathematical}\mspace{14mu}{expression}\mspace{14mu} 6} \right\rbrack\end{matrix}$

Note that the operation was carried out in accordance with the methoddescribed in Pronk, J. T, Steensma, H. Y. and Dijken, J. P., Pyruvate,Metabolism in Saccaromyces cerevisiae. Yeast, 1996, Vol. 12, 1607-1633.

Based on the PDC activity obtained, a pyruvic acid saturation curve wascreated with [V]=PDC enzyme accuracy (U/mg) on the vertical axis and[S]=pyruvic acid concentration (mM) on the horizontal axis. This graphis shown in FIG. 16.

Furthermore, Lineweaver-Burk plots computed from individual inversenumbers were respectively created with 1/[V] and 1/[S] on the verticaland horizontal axes. These graphs are shown in FIG. 17. The pyruvic acidconcentration substrate affinity (Km value) was computed utilizing thefact that the intersection between the Lineweaver-Burk plot and the1/[V] axis is l/Vmax and the intersection between the plot and the 1/[S]axis is −1/Km.

The Lineweaver-Burk plot showed that the pyruvic acid Km value of theyeast-derived pyruvate decarboxylase was 0.346 mM.

This value of 0.346 mM is 3.46 times the pyruvic acid Km value of thebovine-derived L-lactate dehydrogenase, which was 0.1 mM in Embodiment6. In other words, the bovine-derived L-lactate dehydrogenase has 3.46times the substrate affinity of the yeast-derived pyruvatedecarboxylase.

Furthermore, this value of 0.346 mM is 0.346 times the pyruvic acid Kmvalue of the Bifidobacterium longum-derived L-lactate dehydrogenase,which was 1.0 mM. In other words, said Lactobacillus-derived L-lactatedehydrogenase has only 0.346 times the substrate affinity of theyeast-derived pyruvate decarboxylase.

The publications, including the patents and patent applicationspecifications quoted in this Specification, are made part of thisSpecification through their quotation in entirety, as though each ofthese publications were clearly and individually described. Patentapplication specifications in Japanese patent application numbers2001-286637, 2002-128323, 2001-287159, 2002-128286, 2002-65880, and200265879 are also made part of this Specification through theirquotation, as though each of these specifications were clearly andindividually described.

INDUSTRIAL FIELD OF APPLICATION

The present invention can provide a technology for controlling theproduction of ethanol and stably mass-producing lactic acid inside ahost organism having the pyruvate decarboxylase gene.

[Sequence Table Free Text]

SEQ ID NO:3: Modified DNA for coding Lactate dehydrogenase

SEQ ID NO:4: Modified DNA for coding Lactate dehydrogenase

SEQ ID NOs:5 through 39: Primers

1. A bacterial or yeast transformant into which has been incorporated alactate dehydrogenase gene coding sequence, wherein the lactatedehydrogenase coding sequence encodes a foreign protein having lactatedehydrogenase activity and pyruvic acid substrate affinity that equalsor exceeds the pyruvic acid substrate affinity of the pyruvatedecarboxylase inherent in the host organism, wherein a single copy ofthe lactate dehydrogenase gene coding sequence has been incorporatedsuch that it is under the control of a genomic pyruvate decarboxylasegene promoter on the host chromosome, or such that it is under thecontrol of a structural and functional homologue of the genomic pyruvatedecarboxylase gene promoter, which replaces the genomic pyruvatedecarboxylase gene promoter on the host chromosome, and wherein thepyruvate decarboxylase gene on the host chromosome is replaced with thesingle copy of the lactate dehydrogenase gene coding sequence, whereinthe foreign protein is coded by the DNA sequence shown in SEQ ID NO:3.2. The transformant according to claim 1, having the DNA sequence shownin SEQ 1D NO:4 as the DNA sequence for coding the foreign protein.
 3. Atransformant of the Saccharomyces family into which a single copy of alactate dehydrogenase gene coding sequence has been incorporated,wherein the lactate dehydrogenase gene coding sequence encodes abovine-derived lactate dehydrogenase or its homologue and has beenincorporated such that the single copy of the lactate dehydrogenase genecoding sequence is under the control of a genomic pyruvate decarboxylase1 gene promoter on the host chromosome of the Saccharomyces family, orsuch that the single copy of the lactate dehydrogenase gene codingsequence is under the control of a structural and functional homologueof the genomic pyruvate decarboxylase 1 gene promoter, which replacesthe genomic pyruvate decarboxylase 1 gene promoter on the hostchromosome, and wherein the pyruvate decarboxylase 1 gene on the hostchromosome has been replaced with the single copy of the lactatedehydrogenase gene coding sequence encoding the bovine-derived lactatedehydrogenase or its homologue, wherein the bovine-derived lactatedehydrogenase or its homologue is encoded by the DNA sequence shown inSEQ ID NO:3.
 4. The transformant according to claim 3, having the DNAsequence shown in SEQ ID NO:4 as the DNA sequence for encoding thebovine-derived lactate dehydrogenase or its homologue.